Project Summary The increasing emergence of antimicrobial-resistant bacteria/fungi has become a growing global threat due to the misuse and overuse of antimicrobial drugs. In order to combat infections and reduce anti-microbial resistance, it is essential to detect and characterize bacterial/fungal susceptibility to antimicrobial in the early stages of infections to reduce the inappropriate use of antimicrobial drugs and the death rate. To address this urgent medical condition, it is critical to rapidly and accurately determine the antimicrobial susceptibility of bacteria/fungi so that optimal therapy drugs can be prescribed early in the disease process. Conventional methods for antimicrobial susceptibility testing, such as agar plates and broth dilution assays, detect phenotypic resistance based on bacterial/fungal growth in the presence of antimicrobial drugs being tested. A major limitation of these methods is that they are based on culture and require at least 16 to 24 h to conduct. To address this unmet need, a microsecond-scale stimulated Raman spectroscopic imaging platform is proposed to enable in situ detection of a single bacterium in complex environment at sub-micron resolution and early determination of its response to an antimicrobial drug. An interdisciplinary team will conduct the proposed study. Dr. Ji-Xin Cheng (PI) is an inventor and leading expert in coherent Raman scattering microscopy. Dr. Mohamed Seleem (co-PI) is a DVM-scientist with broad expertise in infectious diseases and microbiology. Dr. Ryan F. Relich (consultant), Medical Director of the Indiana University Health Clinical Virology and Serology Laboratories, has extensive experience in clinical diagnosis of infectious diseases. The team?s central hypothesis that microsecond-scale coherent Raman spectroscopic imaging will enable in situ analysis of single microbial cells enriched directly from a clinical sample (whole blood). To test this hypothesis, the team will demonstrate fast determination of antimicrobial response through microsecond-scale stimulated Raman imaging of metabolic activity in a single living bacterium (aim 1), develop a microsecond-scale broadband stimulated Raman spectroscopic microscope for label-free discrimination of bacteria and determination of anti-microbial susceptibility (aim 2), and demonstrate early detection and fast antimicrobial susceptibility profiling of fungal infections (aim 3). The proposed rapid AST method works for bacteria/fungi in complex environment and at the single cell level. Therefore, long-time specimen culture and subculture to get bacterial/fungal isolate can be avoided. The characteristics of this approach offer a significant advancement over current approaches for treatment of bacterial/fungal infections.